1. Field of the Invention
The present invention relates generally to the fields of gene therapy. More particularly, it concerns gene transfer using adeno-associated virus and methods of increasing transcription and promoting replication of transgenes.
2. Description of Related Art
Gene therapy protocols involving recombinant viral vectors are gaining wide attention and have immense potential to become the future mode of molecular medicine. Of the different viral vectors attempted to mediate gene transfer, the retrovirus and adenovirus-based vector systems have been extensively investigated over a decade. Recently, adeno-associated virus (AAV) has emerged as a potential alternative to the more commonly used retroviral and adenoviral vectors (Muzyczka, 1992; Carter, 1992; Flotte and Carter, 1995; Chatterjee et al., 1995; Chatterjee and Wong, 1996). While studies with retroviral and adenoviral mediated gene transfer raise concerns over potential oncogenic properties of the former, and immunogenic problems associated with the latter, AAV has not been associated with any such pathological indications (Berns and Bohenzky, 1987; Berns and Giraud, 1996).
In addition, AAV possesses several unique features that make it more desirable than the other vectors. Unlike retroviruses, AAV can infect non-dividing cells; wild-type AAV has been characterized by integration, in a site-specific manner, into chromosome 19 of human cells (Kotin et al., 1990; Kotin et al., 1991; Samulski et al., 1991), and AAV also possesses anti-oncogenic properties (Berns and Giraud, 1996). Recombinant AAV genomes are constructed by molecularly cloning DNA sequences of interest between the AAV ITRs, eliminating the entire coding sequences of the wild-type AAV genome. The AAV vectors thus produced lack any of the coding sequences of wild-type AAV, yet retain the property of stable chromosomal integration and expression of the recombinant genes upon transduction both in vitro and in vivo (Bertran et al., 1996; Kearns et al., 1996; Ponnazhagan et al., 1997a).
Recent studies have suggested that following infection, the leading strand viral DNA synthesis is a rate-limiting step in the efficient transduction by AAV vectors (Fisher et al., 1996; Ferrari et al., 1996). AAV inverted terminal repeats (ITRs) contain 145 nucleotides (nts) each, the terminal 125 nts of which are palindromic and form T-shaped hairpin (HP) structures, and the 3′-HP structure serves as a primer for AAV DNA replication.
The single-stranded nature of the AAV genome significantly impacts upon the transduction efficiency since the second-strand viral DNA synthesis is the rate-limiting step. It is important to elucidate a factor that is capable of circumventing this step so that AAV vectors may be more effectively used Once such a factor is elucidated, it will be possible to increase the transcription and replication from an adeno-associated viral (AAV) vector.